Selective detection of Bordetella species

ABSTRACT

A process for detecting  Bordetella  spp. nucleic acid in a biological sample includes producing an amplification product(s) by amplifying one or more  Bordetella  spp. in a multiplex single chamber PCR assay, and measuring the amplification product(s) to detect or distinguish  Bordetella  spp. in the biological sample. Also provided are reagents and methods for detecting and distinguishing  Bordetella  spp. from each other and other bacteria or viruses. A kit is provided for detecting and quantifying one or more  Bordetella  spp. in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the United States national stage ofPCT/US2010/032408 filed Apr. 26, 2010, which claims priority of U.S.Provisional Patent Application Ser. No. 61/172,382 filed Apr. 24, 2009,which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to processes for detection of bacteriain fluid samples, and in particular to selective detection of Bordetellasp. in biological or other fluid media. Processes are described forrapid, sensitive, and specific multiplex detection and differentiationof B. pertussis, B. parapertussis, and B. holmesii in human and animalbiological samples and quantification thereof. Diagnostic kits areprovided for detection and differentiation of Bordetella sp. in aclinical, laboratory, or field setting.

BACKGROUND OF THE INVENTION

Bordetella pertussis, the etiologic agent of whooping cough, is asubstantial cause of morbidity and infant mortality worldwide. Themajority of the estimated annual 279,000 infant pertussis deaths occurin developing countries (WHO, 2005). In the United States, 25,616 casesof pertussis were reported during 2005 (Kretsinger et al., MMWR Recomm.Rep. 55:1-44, 2006). The three most common Bordetella human upperrespiratory pathogens are B. pertussis, B. parapertussis, and B.holmesii. Others of the nine known species include Bordetellabronchiseptica, Bordetella hinzii, and Bordetella trematum. The B.parapertussis infection is less severe than B. pertussis, with symptomsthat include a paroxysmal cough and a whoop that, if present, persistsfor a shorter duration (Mattoo and Cherry, Clin. Microbiol. Rev.18:326-382, 2005). B. holmesii was first associated with septicemia inimmunocompromised patients (Weyant et al., J. Clin. Microbiol. 33:1-7,1995) and has been recovered from nasopharyngeal specimens of patientswith pertussis-like diseases in Massachusetts (Yih et al., Emerg.Infect. Dis. 5:441-443, 1999). Therefore, differentiating these threespecies is essential to providing an accurate diagnosis ofpertussis-like diseases or whooping cough in patient populations.

Prior art diagnostic methods to detect B. pertussis include culture,singleplex real-time polymerase chain reaction (PCR), and serology.Culture is 100% specific; however, it suffers from low sensitivity(12-60%) and lengthy incubation periods from 5 to 10 days (Wendelboe andVan Rie, Expert Rev. Mol. Diagn. 6:857-864, 2006). The sensitivity ofculture is highest in young infants tested after a short duration ofsymptoms and the lowest in adolescents and adults tested after a longerduration of symptoms (Riffelmann et al., J. Clin. Microbiol.43:4925-4929, 2005).

In the United States, the number of reported adult and adolescentpertussis cases has substantially increased in the last two decades;however, most cases are not laboratory confirmed, highlighting the needfor additional diagnostic tests with higher sensitivity than culture.Although the lengthy incubation period for culture makes it impracticalas a rapid diagnostic test, culture in combination with PCR is areliable option for case detection during a public health response to apertussis outbreak (Sotir et al., Clin. Infect. Dis. 44:1216-1219,2007). Serologic assays are reviewed in detail elsewhere (Broder et al.,2006; Centers for Disease Control and Prevention [CDC], MMWR 56:837-842,2007; Fry et al., J. Med. Microbiol. 53:519-525, 2004; Kretsinger etal., 2006; Mattoo and Cherry, 2005; Sotir et al., 2007).

Real-time PCR assays, which can be rapid (2-24 h), specific (86-100%),and sensitive (70-99%), have been designed to detect B. pertussis(Wendelboe and Van Rie, 2006). Conventional PCR assays introduced in1989 (Houard et al., Res. Microbiol. 140:477-487, 1989) for thedetection of B. pertussis are being replaced by real-time PCR assays inclinical diagnostics. However, standardizing these PCR methods ischallenging (Arber, FEMS Microbiol. Rev. 24:1-7, 2000; Broder et al.,MMWR Recomm. Rep., 55:1-50, 2006; CDC, MMWR 53:216-219, 2004; Douglas etal., J Med Microbiol 38:140-144, 1993; Fry et al., 2004). Severalchromosomal regions have been used as targets, such as adenylate cyclase(Douglas et al., 1993), pertactin (Byrne and Slack, BMC Infect. Dis.6:53-61, 2006; Makinen et al., Emerg. Infect. Dis. 7:952-958, 2001;Vincart et al., J. Med. Microbiol. 42:847-849, 2007), porin (Li et al.,J. Clin. Microbiol. 32:783-789, 1994; Qin et al., J. Clin. Microbiol.45:506-511, 2007), recA (Antila et al., J. Med. Microbiol. 55:1043-1051,2006; Qin et al., 2007; Vielemeyer et al., J. Clin. Microbiol.42:847-849, 2004), pertussis toxin promoter region (Houard et al., Res.Microbiol. 140:477-487, 1989; Knorr et al., BMC Infec. Dis. 6:62-74,2006; Makinen et al., 2001; Nygren et al., J. Clin. Microbiol. 38:55-60,2000; Reizenstein, Dev. Biol. Stand. 89:247-254, 1997; Stefanelli etal., Diag. Microbiol. Infect. Dis. 24:197-200, 1996), and, mostfrequently, insertion sequence IS481 (Glare et al., J. Clin. Microbiol.28:1982-1987, 1990; McPheat and McNally, J. Gen. Microbiol. 133:323-330,1987a, FEMS Microbiol. Lett. 41:357-360, 1987b; Templeton et al., J.Clin. Microbiol. 41:4121-4126, 2003; van der Zee et al., J. Clin.Microbiol. 31:2134-2140, 1993).

Accurate identification of pertussis cases in respiratory diseaseoutbreaks is of particular importance because some interventions, suchas vaccination, are pathogen specific. PCR confirmation is included inthe case definition of pertussis from the Council of State andTerritorial Epidemiologists. A confirmed case of pertussis is any personwith acute cough illness of any duration with isolation of B. pertussis,or a case that meets the clinical case definition and is confirmed byPCR, or a case that meets the clinical definition and isepidemiologically linked directly to a case confirmed by either cultureor PCR (Broder et al., 2006; Kretsinger et al., 2006). However, inrespiratory disease outbreaks, positive results with a single PCR assaytargeting the insertion sequence IS481 have led to a false diagnosis ofpertussis, demonstrating the need for different or additional target(CDC, 2007; Farrell et al., J. Clin. Microbiol. 38:4499-4502, 2000;Lievano et al., J. Clin. Microbiol. 40:2801-2805, 2002; Muyldermans etal., J. Clin. Microbiol. 43:30-35, 2005).

Thus, there is a need for a rapid, sensitive, and discriminatory assayfor detection of Bordetella sp. in complex clinical or laboratorysamples in the presence or absence of other bacterial or viral agents.

SUMMARY OF THE INVENTION

A composition including an isolated nucleotide sequence of one or moreof SEQ ID NOS: 1-12 or modifications of any thereof is providedherewith. The composition is operative to provide a rapid, sensitive,and selective detection of Bordetella species. The composition is usefulas a uniplex or multiplex detection test. A kit is provided with a setof primers and a probe for specific detection of B. pertussis, B.holmesii, B. parapertussis, or sensitive to B. pertussis/B.parapertussis yet insensitive to B. holmesii.

A kit is provided to perform a process of detection and distinguishmentbetween one or more Bordetella species in a biological sample thatincludes exposing the sample to forward primers of SEQ ID NOS: 1, 4 and7 as well as reverse primers of SEQ ID NO: 2, 5 or 8 under conditionssuitable for polymerase chain reaction to measure respectiveamplification products therewith indicative of B. pertussis, B. holmesiior B. parapertussis, respectively. Such a process is amenable tooperation in a multiplex mode. The kit and the process it performsprovides rapid diagnosis of Bordetella infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are assay plots for IS481 (A) and PtxS1 (B) sequences,both showing linear application over orders of magnitude; and

FIG. 2 is a plot of a multiplex assay for different strains of B.pertussis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The high incidence of false diagnosis of Bordetella infection combinedwith the highly species specific vaccines available makes diagnosing anddistinguishing Bordetella species in suspect patients paramount forcontrolling outbreaks. The instant invention has utility for detectionand distinguishing of Bordetella species in biological samples,diagnosis of disease associated therewith, and discrimination againstother bacterial and viral pathogens. It is appreciated that an inventiveprocess is readily performed for financial compensation.

The following definitional terms are used throughout the specificationwithout regard to placement relative to these terms.

As used herein, the term “variant” defines either a naturally occurringgenetic mutant of Bordetella or a recombinantly prepared variation ofBordetella, each of which contain one or more mutations in its genomecompared to the Bordetella of B. pertussis, B. parapertussis, or B.holmesii. The term “variant” may also refer to either a naturallyoccurring variation of a given peptide or a recombinantly preparedvariation of a given peptide or protein in which one or more amino acidresidues have been modified by amino acid substitution, addition, ordeletion.

As used herein, the term “analog” in the context of a non-proteinaceousanalog defines a second organic or inorganic molecule that possesses asimilar or identical function as a first organic or inorganic moleculeand is structurally similar to the first organic or inorganic molecule.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative defines a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated. Aderivative also defined as a degenerate base mimicking a C/T mix such asthat from Glen Research Corporation, Sterling, Va., illustrativelyLNA-dA or LNA-dT, or other nucleotide modification known in the art orotherwise.

As used herein, the term “mutant” defines the presence of mutations inthe nucleotide sequence of an organism as compared to a wild-typeorganism.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing under whichnucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to each other typicallyremain hybridized to each other. Such hybridization conditions aredescribed in, for example but not limited to, Current Protocols inMolecular Biology, John Wiley & Sons, NY (1989), 6.3.1 6.3.6.; BasicMethods in Molecular Biology, Elsevier Science Publishing Co., Inc., NY(1986), pp. 75 78, and 84 87; and Molecular Cloning, Cold Spring HarborLaboratory, NY (1982), pp. 387 389, and are well known to those skilledin the art. A preferred, non-limiting example of stringent hybridizationconditions is hybridization in 6× sodium chloride/sodium citrate (SSC),0.5% SDS at about 68° C. followed by one or more washes in 2×SSC, 0.5%SDS at room temperature. Another preferred, non-limiting example ofstringent hybridization conditions is hybridization in 6×SSC at about45° C. followed by one or more washes in 0.2×SSC, 0.1% SDS at 50 to 65°C.

An “isolated” or “purified” nucleotide or oligonucleotide sequence issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the nucleotide is derived, oris substantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of a nucleotide/oligonucleotide in whichthe nucleotide/oligonucleotide is separated from cellular components ofthe cells from which it is isolated or produced. Thus, anucleotide/oligonucleotide that is substantially free of cellularmaterial includes preparations of the nucleotide having less than about30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of contaminatingmaterial. When nucleotide/oligonucleotide is produced by chemicalsynthesis, it is preferably substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the nucleotide/oligonucleotide haveless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or compounds other than the nucleotide/oligonucleotide ofinterest. In a preferred embodiment of the present invention, thenucleotide/oligonucleotide is isolated or purified.

As used herein, the term “isolated” bacteria is one which is separatedfrom other organisms which are present in the natural source of thebacteria, e.g., biological material such as cells, blood, serum, plasma,saliva, urine, stool, sputum, nasopharyngeal aspirates, and so forth.The isolated bacteria can optionally be used to infect a subject cell.

As used herein, the term “biological sample” is defined as sampleobtained from a biological organism, a tissue, cell, cell culturemedium, or any medium suitable for mimicking biological conditions, orfrom the environment. Non-limiting examples include saliva, gingivalsecretions, cerebrospinal fluid, gastrointestinal fluid, mucous,urogenital secretions, synovial fluid, blood, serum, plasma, urine,cystic fluid, lymph fluid, ascites, pleural effusion, interstitialfluid, intracellular fluid, ocular fluids, seminal fluid, mammarysecretions, vitreal fluid, and nasal secretions, throat or nasalmaterials. In a preferred embodiment, bacterial agents are contained inserum, whole blood, nasopharyngeal fluid, throat fluid, or otherrespiratory fluid.

As used herein, the term “medium” refers to any liquid or fluidbiological sample in the presence or absence of bacteria. Non-limitingexamples include buffered saline solution, cell culture medium,acetonitrile, trifluoroacetic acid, combinations thereof, or any otherfluid recognized in the art as suitable for combination with bacteria orother cells, or for dilution of a biological sample or amplificationproduct for analysis.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first amino acid or nucleic acidsequence for optimal alignment with a second amino acid or nucleic acidsequence). The nucleotides at corresponding nucleotide positions arethen compared. When a position in the first sequence is occupied by thesame nucleotide as the corresponding position in the second sequence,then the molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=number of identicaloverlapping positions/total number of positions×100%). In oneembodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:22642268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searchesare performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules of the present invention. BLAST proteinsearches are performed with the XBLAST program parameters set, e.g., toscore 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST are utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST is used to perform an iterated search which detects distantrelationships between molecules (Id.). When utilizing BLAST, GappedBLAST, and PSI Blast programs, the default parameters of the respectiveprograms (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBIwebsite). Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, 1988, CABIOS 4:11 17. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 is used.

The percent identity between two sequences is determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the terms “subject” and “patient” are synonymous andrefer to a human or non-human animal, preferably a mammal including ahuman, non-primate such as cows, pigs, horses, goats, sheep, cats, dogs,avian species and rodents; and a non-human primate such as monkeys,chimpanzees, and apes; and a human, also denoted specifically as a“human subject”.

The instant inventive process provides a rapid, specific, and sensitiveassay process for detection of Bordetella spp. in biological samples byamplifying one or more nucleotide sequences that allow an investigatorto distinguish between species and are present in a biological sample byprocesses similar to the polymerase chain reaction (PCR). The inventionis preferably used to distinguish B. pertussis, B. parapertussis, and B.holmesii.

A family of forward and reverse nucleotide primer pairs is provided thateach amplify a portion of one or more species of Bordetella. In theinventive process an oligonucleotide forward primer with a nucleotidesequence complementary to a unique sequence in a Bordetella nucleotidesequence, illustratively in the IS481 sequence (GenBank Accession No.M28220), is hybridized to its complementary sequence and extended.Similarly, a reverse oligonucleotide primer complementary to a secondstrand of Bordetella DNA in the same or an alternate region ishybridized and extended. This system allows for amplification ofspecific nucleotide sequences and is operable for simultaneous orsequential detection systems.

The present invention relates to the use of sequence information ofBordetella for diagnostic processes. More particularly, the presentinvention provides a process for detecting the presence or absence ofnucleic acid molecules of one or more Bordetella species, natural orartificial variants, analogs, or derivatives thereof, in a biologicalsample. The process involves obtaining a biological sample from one ormore various sources and contacting the sample with a compound or anagent capable of detecting a nucleic acid sequence of Bordetella spp.,natural or artificial variants, analogs, or derivatives thereof, suchthat the presence of Bordetella, natural or artificial variants,analogs, or derivatives thereof, is detected in the sample. In apreferred embodiment, the presence of B. pertussis, B. holmesii, or B.parapertussis, natural or artificial variants, analogs, or derivativesthereof, is detected in the sample by a real-time polymerase chainreaction (real-time PCR) using primers that are constructed based on apartial nucleotide sequence of the B. pertussis genome.

Preferably, detection of one or more species of Bordetella isaccomplished by amplification of particular nucleotide sequencesspecific for one of B. pertussis, B. holmesii, or B. parapertussis. Morepreferably, a region specific to B. pertussis and B. holmesii is theIS481 sequence which encompasses nucleotides 862-927 of GenBankAccession No. M28220. A nucleotide sequence specific for B. holmesii ishIS1001 sequence which encompasses nucleotides 41-107 of GenBankAccession No. AY786982. A nucleotide sequence specific for B.parapertussis is pIS1001 sequence which encompasses nucleotides 135-199of GenBank Accession No. X66858. A nucleotide sequence specific for B.parapertussis and B. pertussis but is not detectable in B. holmesii isptxS1 sequence which encompasses the sequence of GenBank Accession No.M14378.

In a preferred embodiment, a forward primer to be used in a real-timePCR process is 5′-CAAGGCCGAACGCTTCAT-3′ (SEQ ID NO: 1), and a reverseprimer 5′-GAGTTCTGGTAGGTGTGAGCGTAA-3′ (SEQ ID NO: 2). Illustratively,the forward primer and reverse primer are used to amplify a region ofBordetella nucleotide sequence to produce a first amplification product.

A preferred agent for detecting Bordetella spp. nucleic acid sequences,or a first amplification product produced therefrom, is a labelednucleic acid probe capable of hybridizing thereto or to amplificationproducts produced by PCR amplification of a region between a forward andreverse primer pair. In the above preferred embodiment, the nucleic acidprobe is a nucleic acid molecule comprising or consisting of the nucleicacid sequence of 5′-CAGTCGGCCTTGCGTGAGTGGG-3′ (SEQ ID NO: 3), whichsufficiently specifically hybridizes under stringent conditions to a B.pertussis nucleic acid sequence.

Preferably, the inventive process is operable to distinguish or detectone or more species of Bordetella. Illustratively, the inventive processis operable to detect one or more of B. pertussis, B. parapertussis, andB. holmesii and identify whether one or more of these or otherBordetella strains are present in a biological sample.

B. holmesii is illustratively detected using a forward primer of5′-GGCGACAGCGAGACAGAATC-3′ (SEQ ID NO: 4) and reverse primer of5′-GCCGCCTTGGCTCACTT-3′ (SEQ ID NO: 5). Surprisingly, this primer pairwill amplify the hIS1001 region of B. holmesii and is specific for thisstrain demonstrating no amplification of B. pertussis or B.parapertussis. The IS1001 insertion sequence is a 1,306 bp sequencecontaining inverted repeats at its termini. (van der Dee, A, et al., J.Bacteriol., 1993; 75:141-147.) IS1001 was identified as an insertionelement in B. parapertussis and has been used to detect the presence ofB. parapertussis in biological samples to distinguish B. parapertussisfrom B. pertussis. Id. The hIS1001 sequence encompasses the sequence ofGenBank Accession No. AY786982. The region amplified by primers SEQ IDNOS: 4 and 5 reveals that this insertion is present in B. holmesii andthe sequence amplified is specific only to B. holmesii.

Detection of the preferred amplification product of SEQ ID NOS: 4 and 5is accomplished by hybridizing a non-degenerate probe complementary tothe amplification product. Illustratively, a probe operable is5′-CGTGCAGATAGGCTTTTAGCTTGAGCGC-3′(SEQ ID NO: 6). It is appreciated thatall probes used herein are optionally modified, or replaced by adifferent sequence capable to detecting an amplification product withinthe target amplification product sequence.

A third species of Bordetella, B. parapertussis, is optionally detectedor distinguished in the inventive process. The insertion element pIS1001(GenBank Accession No. X66858) is preferably amplified by forward primer5′-TCGAACGCGTGGAATGG-3′ (SEQ ID NO: 7) and reverse primer5′-GGCCGTTGGCTTCAAATAGA-3′ (SEQ ID NO: 8). The amplification product ofthis region of the IS1001 sequence is specific to B. parapertussis. Thisamplification product is preferably detected by a non-degenerate probe.Preferably the non-degenerate probe is 5′-AGACCCAGGGCGCACGCTGTC-3′ (SEQID NO: 9).

Primer and probe sequences most preferred in the subject invention areillustrated in Table 1.

TABLE 1 Sequences of primers and probes Amplicon Length TargetPrimer/Probe Sequence 5′→3′ (bp) IS481^(a) 825U18CAAGGCCGAACGCTTCAT (SEQ ID NO: 1) 66 bp 894L24GAGTTCTGGTAGGTGTGAGCGTAA (SEQ ID NO: 2) 871U22P^(b)CAGTCGGCCTTGCGTGAGTGGG (SEQ ID NO: 3) hIS1001^(c) BHIS41U20GGCGACAGCGAGACAGAATC (SEQ ID NO: 4) 67 bp BHIS91L17GCCGCCTTGGCTCACTT (SEQ ID NO: 5) BHIS62U28P^(d) CGTGCAGATAGGCTTTTAGCTTGAGCGC ((SEQ ID NO: 6) pIS1001^(e) 135U17TCGAACGCGTGGAATGG (SEQ ID NO: 7) 65 bp 199L20GGCCGTTGGCTTCAAATAGA (SEQ ID NO: 8) 157U21PHEX^(f)AGACCCAGGGCGCACGCTGTC (SEQ ID NO: 9) ptxS1^(g) 402U16CGCCAGCTCGTACTTC (SEQ ID NO: 10) 55 bp 422L15GATACGGCCGGCATT (SEQ ID NO: 11) 419U22P^(b)AATACGTCGACACTTATGGCGA (SEQ ID NO: 12) ^(a)Accession no. M28220.^(b)Probe 5′ end labeled with 6-carboxyfluorescein FAM™ and 3′ endlabeled with Black Hole Quencher® 1 (BHQ1). ^(c)Accession no. AY786982.^(d)Probe 5′ end labeled with Quasar 670 and 3′ end labeled with BlackHole Quencher® 2 (BHQ2). ^(e)Accession no. X66858. ^(f)Probe 5′ endlabeled with HEX™ and 3′ end labeled with Black Hole Quencher® 1 (BHQ1).^(g)Accession no. M14378

The IS481 assay targets a region downstream from the inverted repeatgenerating a 66-bp amplicon, and the ptxS1 assay targets a regionapproximately 400 bp downstream of the start codon of the pertussistoxin subunit 1, the ptxA gene, generating a 55-bp amplicon.

The process of the present invention can involve a real-timequantitative PCR assay. In a preferred embodiment, the quantitative PCRused in the present invention is TaqMan assay (Holland et al., PNAS88(16):7276 (1991)). It is appreciated that the current invention isamenable to performance on other real-time PCR systems and protocolsthat use alternative reagents illustratively including, but not limitedto Molecular Beacons probes, Scorpion probes, multiple reporters formultiplex PCR, combinations thereof, or other DNA detection systems.

The assays are performed on an instrument designed to perform suchassays, for example those available from Applied Biosystems (FosterCity, Calif.). In more preferred specific embodiments, the presentinvention provides a real-time quantitative PCR assay to detect thepresence of one or more Bordetella species, natural or artificialvariants, analogs, or derivatives thereof, in a biological sample bysubjecting the Bordetella nucleic acid from the sample to PCR reactionsusing specific primers, and detecting the amplified product using aprobe. In preferred embodiments, the probe is a TaqMan® probe whichconsists of an oligonucleotide with a 5′-reporter dye and a 3′-quencherdye.

A fluorescent reporter dye, such as FAM™ dye (illustratively6-carboxyfluorescein), is covalently linked to the 5′ end of theoligonucleotide probe. Other dyes illustratively include TAMRA™,AlexaFluor™ dyes such as AlexaFluor™ 495 or 590, Cascade Blue®, MarineBlue®, Pacific Blue®, Oregon Green®, Rhodamine, Fluoroscein, TET™, HEX™,Cy5™,Cy3™, Quasar670, and Tetramethylrhodamine. Each of the reporters isquenched by a dye at the 3′ end or other non-fluorescent quencher.Quenching molecules are suitably matched to the fluorescence maximum ofthe dye. Any suitable fluorescent probe for use in real-time PCRdetection systems is illustratively operable in the instant invention.Similarly, any quenching molecule for use in real-time PCR systems isillustratively operable. In a preferred embodiment a6-carboxyfluorescein reporter dye is present at the 5′-end and matchedto BLACK HOLE QUENCHER® (BHQ1, Biosearch Technologies, Inc., Novato,Calif.). The fluorescence signals from these reactions are captured atthe end of extension steps as PCR product is generated over a range ofthe thermal cycles, thereby allowing the quantitative determination ofthe bacterial load in the sample based on an amplification plot.

The Bordetella nucleic acid sequences are optionally amplified beforebeing detected. The term “amplified” defines the process of makingmultiple copies of the nucleic acid from a single or lower copy numberof nucleic acid sequence molecule. The amplification of nucleic acidsequences is carried out in vitro by biochemical processes known tothose of skill in the art. The amplification agent may be any compoundor system that will function to accomplish the synthesis of primerextension products, including enzymes. Suitable enzymes for this purposeinclude, for example, E. coli DNA polymerase I, Taq polymerase, Klenowfragment of E. coli DNA polymerase I, T4 DNA polymerase, AmpliTaq GoldDNA Polymerase from Applied Biosystems, other available DNA polymerases,reverse transcriptase (preferably iScript RNase H+ reversetranscriptase), ligase, and other enzymes, including heat-stable enzymes(i.e., those enzymes that perform primer extension after being subjectedto temperatures sufficiently elevated to cause denaturation). In apreferred embodiment, the enzyme is hot-start iTaq DNA polymerase fromBio-rad (Hercules, Calif.). Suitable enzymes will facilitate combinationof the nucleotides in the proper manner to form the primer extensionproducts that are complementary to each mutant nucleotide strand.Generally, the synthesis is initiated at the 3′-end of each primer andproceed in the 5′-direction along the template strand, until synthesisterminates, producing molecules of different lengths. There may beamplification agents, however, that initiate synthesis at the 5′-end andproceed in the other direction, using the same process as describedabove. In any event, the process of the invention is not to be limitedto the embodiments of amplification described herein.

One process of in vitro amplification, which is used according to thisinvention, is the polymerase chain reaction (PCR) described in U.S. Pat.Nos. 4,683,202 and 4,683,195. The term “polymerase chain reaction”refers to a process for amplifying a DNA base sequence using aheat-stable DNA polymerase and two oligonucleotide primers, onecomplementary to the (+)-strand at one end of the sequence to beamplified and the other complementary to the (−)-strand at the otherend. Because the newly synthesized DNA strands can subsequently serve asadditional templates for the same primer sequences, successive rounds ofprimer annealing, strand elongation, and dissociation produce rapid andhighly specific amplification of the desired sequence. Many polymerasechain processes are known to those of skill in the art and may be usedin the process of the invention. For example, DNA is subjected to 30 to35 cycles of amplification in a thermocycler as follows: 95° C. for 30sec, 52 to 60° C. for 1 min, and 72° C. for 1 min, with a finalextension step of 72° C. for 5 min. For another example, DNA issubjected to 35 polymerase chain reaction cycles in a thermocycler at adenaturing temperature of 95° C. for 30 sec, followed by varyingannealing temperatures ranging from 54 to 58° C. for 1 min, an extensionstep at 70° C. for 1 min, with a final extension step at 70° C. for 5min. The parameters of PCR cycling times and number of steps aredependent on the primer pair, their melting temperature, and otherconsiderations obvious to those known in the art. It is appreciated thatoptimizing PCR parameters for various probe sets is well within theskill of the art and is performed as mere routine optimization.

The primers for use in amplifying the nucleic acid sequences ofBordetella may be prepared using any suitable process, such asconventional phosphotriester and phosphodiester processes or automatedembodiments thereof so long as the primers are capable of hybridizing tothe nucleic acid sequences of interest. One process for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primergenerally must prime the synthesis of extension products in the presenceof the inducing agent for amplification.

Primers used according to the process of the invention are complementaryto each strand of nucleotide sequence to be amplified. The term“complementary” means that the primers must hybridize with theirrespective strands under conditions, which allow the agent forpolymerization to function. In other words, the primers that arecomplementary to the flanking sequences hybridize with the flankingsequences and permit amplification of the nucleotide sequence.Preferably, the 3′ terminus of the primer that is extended is perfectlybase paired with the complementary flanking strand. Preferably, probespossess nucleotide sequences complementary to one or more strands of theamplification product such as from IS481, hIS1001, or pIS1001. Morepreferably, the primers are complementary to genetic sequences of B.pertussis IS481 insert as illustrated in Accession No. M28220; B.holmesii as illustrated in Accession No. AY786982; and for B.parapertussis as illustrated in Accession No. X66858. Most preferably,primers contain the nucleotide sequences of SEQ ID NOS: 1 and 2; 4 and5; and 7 and 8. It is appreciated that the complement of theaforementioned primer and probe sequences are similarly suitable for usein the instant invention. It is further appreciated that oligonucleotidesequences that hybridize with the inventive primer and probes are alsosimilarly suitable. Finally, multiple positions are available forhybridization on the Bordetella genome and will be also suitablehybridization with a probe when used with the proper forward and reverseprimers.

Those of ordinary skill in the art will know of various amplificationprocesses that can also be utilized to increase the copy number oftarget nucleic acid sequence. The nucleic acid sequences detected in theprocess of the invention are optionally further evaluated, detected,cloned, sequenced, and the like, either in solution or after binding toa solid support, by any process usually applied to the detection of aspecific nucleic acid sequence such as another polymerase chainreaction, oligomer restriction (Saiki et al., BioTechnology 3:1008 1012(1985)), allele-specific oligonucleotide (ASO) probe analysis (Conner etal., PNAS 80: 278 (1983)), oligonucleotide ligation assays (OLAs)(Landegren et al., Science 241:1077 (1988)), RNase Protection Assay andthe like. Molecular techniques for DNA analysis have been reviewed(Landegren et al., Science 242:229 237 (1988)). Following DNAamplification, the reaction product may be detected by Southern blotanalysis, with or without using radioactive probes. In such a process,for example, a small sample of DNA containing the nucleic acid sequenceobtained from the tissue or subject is amplified, and analyzed via aSouthern blotting technique. The use of non-radioactive probes or labelsis facilitated by the high level of the amplified signal. In oneembodiment of the invention, one nucleoside triphosphate isradioactively labeled, thereby allowing direct visualization of theamplification product by autoradiography. In another embodiment,amplification primers are fluorescently labeled and run through anelectrophoresis system. Visualization of amplified products is by laserdetection followed by computer assisted graphic display, without aradioactive signal.

Other methods of detecting amplified oligonucleotide illustrativelyinclude gel electrophoresis, mass spectrometry, liquid chromatography,fluorescence, luminescence, gel mobility shift assay, fluorescenceresonance energy transfer, nucleotide sequencing, enzyme-linkedimmunoadsorbent assay, affinity chromatography, chromatography,immunoenzymatic methods (Ortiz, A and Ritter, E, Nucleic Acids Res.,1996; 24:3280-3281), streptavidin-conjugated enzymes, DNA branchmigration (Lishanski, A, et al., Nucleic Acids Res., 2000; 28(9):e42),enzyme digestion (U.S. Pat. No. 5,580,730), colorimetric methods (Lee,K, Biotechnology Letters, 2003; 25:1739-1742), or combinations thereof.

The term “labeled” with regard to the probe is intended to encompassdirect labeling of the probe by coupling (i.e., physically linking) adetectable substance to the probe, as well as indirect labeling of theprobe by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a probe using afluorescently labeled antibody and end-labeling or centrally labeling ofa DNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The detection method of the invention can be usedto detect RNA (particularly mRNA) or genomic nucleic acid in a sample invitro as well as in vivo. For example, in vitro techniques for detectionof nucleic acid include northern hybridizations, in situ hybridizations,RT-PCR, real-time PCR, and DNase protection. Furthermore, in vivotechniques for detection of Bordetella include introducing into asubject organism a labeled antibody directed against a membrane or otherpolypeptide component or directed against a particular nucleic acidsequence of Bordetella. For example, the antibody can be labeled with aradioactive marker whose presence and location in the subject organismcan be detected by standard imaging techniques, includingautoradiography.

The size of the primers used to amplify a portion of the nucleic acidsequence of Bordetella is at optionally least 5, and often 10, 15, 20,25, or 30 nucleotides in length. Preferably, the GC ratio should beabove 30%, 35%, 40%, 45%, 50%, 55%, or 60% so as to prevent hair-pinstructure on the primer. Furthermore, the amplicon should besufficiently long enough to be detected by standard molecular biologymethodologies. The forward primer is preferably shorter than the reverseprimer. Techniques for modifying the T_(m) of either primer are operableherein. An illustrative forward or reverse primer contains LNA-dA andLNA-dT (Glen Research Corporation) so as to match T_(m) with acorresponding alternate primer.

An inventive process uses a polymerization reaction which employs anucleic acid polymerizing enzyme, illustratively a DNA polymerase, RNApolymerase, reverse transcriptase, or mixtures thereof. It is furtherappreciated that accessory proteins or molecules are present to form thereplication machinery. In a preferred embodiment the polymerizing enzymeis a thermostable polymerase or thermodegradable polymerase. Use ofthermostable polymerases is well known in the art such as Taq polymeraseavailable from Invitrogen Corporation. Thermostable polymerases allow apolymerization reaction to be initiated or shut down by changing thetemperature or other condition in the reaction mixture withoutdestroying activity of the polymerase.

Accuracy of the base pairing in the preferred embodiment of DNAsequencing is provided by the specificity of the enzyme. Error rates forTaq polymerase tend to be false base incorporation of 10⁻⁵ or less.(Johnson, Annual Reviews of Biochemistry, 1993: 62:685-713; Kunkel,Journal of Biological Chemistry, 1992; 267:18251-18254). Specificexamples of thermostable polymerases illustratively include thoseisolated from Thermus aquaticus, Thermus thermophilus, Pyrococcuswoesei, Pyrococcus furiosus, Thermococcus litoralis and Thermotogamaritima. Thermodegradable polymerases illustratively include E. coliDNA polymerase, the Klenow fragment of E. coli DNA polymerase, T4 DNApolymerase, T7 DNA polymerase and other examples known in the art. It isrecognized in the art that other polymerizing enzymes are similarlysuitable illustratively including E. coli, T7, T3, SP6 RNA polymerasesand AMV, M-MLV, and HIV reverse transcriptases.

The polymerases are optionally bound to the primer. When the geneticmaterial of Bordetella is a single-stranded DNA molecule due to heatdenaturing the polymerase is bound at the primed end of thesingle-stranded nucleic acid at an origin of replication. A binding sitefor a suitable polymerase is optionally created by an accessory proteinor by any primed single-stranded nucleic acid.

In a further embodiment detection of PCR products is achieved by massspectrometry. Mass spectrometry has several advantages over real-timePCR systems in that it can be used to simultaneously detect the presenceof Bordetella and decipher mutations in target nucleic acid sequencesallowing identification and monitoring of emerging strains. Further,mass spectrometers are prevalent in the clinical laboratory. Similar tofluorescence based detection systems mass spectrometry is capable ofsimultaneously detecting multiple amplification products for amultiplexed and controlled approach to accurately quantifying componentsof biological or environmental samples.

Multiple mass spectrometry platforms are suitable for use in the instantinvention illustratively including matrix assisted laser desorptionionization time of flight mass spectrometry (MALDI), electrospray massspectrometry, electrospray ionization-Fourier transform ion cyclotronresonance mass spectrometry (ESI-FTICR), multi-stage mass spectrometryfragmentation analysis (MS/MS), mass spectrometry coupled with liquidchromatography such as high performance liquid chromatography massspectrometry (HPLC) and ultra performance liquid chromatography isotopedilution tandem mass spectrometry (HPLC-ID/MS/MS), and variationsthereof.

It is appreciated that numerous other detection processes are similarlysuitable for measuring an amplification product by detecting a detectionsignal. Illustrative examples include, but are not limited to, liquidchromatography, mass spectrometry, liquid chromatography/massspectrometry, static fluorescence, dynamic fluorescence, highperformance liquid chromatography, ultra-high performance liquidchromatography, enzyme-linked immunoadsorbent assay, real-time PCR, gelelectrophoresis, or combinations thereof.

Preferably, PCR amplification products are generated using complementaryforward and reverse oligonucleotide primers. In a non-limiting example,Bordetella genetic sequences or fragments thereof are amplified by theprimer pair SEQ ID NOS: 1 and 2 that amplify a conserved sequence in theIS481 insertion sequence and is useful to detect B. pertussis and/or B.holmesii. The resulting amplification product is processed and preparedfor detection by processes known in the art. It is appreciated that thecomplements of SEQ ID NOS: 1 and 2 are similarly suitable for use in theinstant invention. It is further appreciated that oligonucleotidesequences that hybridize with SEQ ID NO: 1 or 2 are also similarlysuitable. Finally, multiple positions are available for hybridization onthe Bordetella genome and will be also suitable hybridization withforward and reverse primers that may or may not be used with a probe forreal-time PCR.

Optionally, multiple amplification products are simultaneously producedin a PCR reaction that is then available for simultaneous detection andquantification. Thus, multiple detection signals are inherently producedor emitted that are separately and uniquely detected in one or moredetection systems. It is appreciated that multiple detection signals areoptionally produced in parallel. Preferably, a single biological sampleis subjected to analysis for the simultaneous or sequential detection ofBordetella genetic sequences. It is appreciated that three or moreindependent or overlapping sequences are simultaneously or sequentiallymeasured in the instant inventive process. Oligonucleotide matchedprimers (illustratively SEQ ID NOS: 1 and 2) are simultaneously orsequentially added and the biological sample is subjected to properthermocycling reaction parameters. For detection by mass spectrometry asingle sample of the amplification products from each gene aresimultaneously analyzed allowing for rapid and accurate determination ofthe presence of Bordetella. Optionally, analysis by real-time PCR isemployed capitalizing on multiple probes with unique fluorescentsignatures. Thus, each gene or other genetic sequence is detectedwithout interference by other amplification products. This multi-targetapproach increases confidence in quantification and provides foradditional internal control.

In a preferred embodiment a triplex approach is used for thesimultaneous detection and distinguishing of three Bordetella species.Preferably, B. pertussis, B. parapertussis, and B. holmesii are eachdetectable in a single biological sample simultaneously. Each real-timePCR assay normally takes approximately 90-100 minutes to run on theinstrument. Combining three assays into one reduces the run time in apreferred embodiment from 300 minutes to 100 minutes. Similarly,utilizing the multiplex assay is cost effective. Instead of using mastermixes, plastics, etc. for three individual assays, only one assay is runsuch that the cost is approximately ⅓ that of three assays.

Illustratively, three sets of matched primer pairs are used. Preferably,SEQ ID NOS: 1 and 2 are operable for amplification of nucleotidesequences present in B. pertussis and possibly B. holmesii. SEQ ID NOS:4 and 5 are operable for amplification of nucleotide sequences presentin and specific for B. holmesii. SEQ ID NOS: 7 and 8 are foramplification of nucleotide sequences present in and specific for B.parapertussis. Thus, simultaneous presence of all three primer pairs ina single reaction chamber will produce first, second, and thirdamplification products in a biological sample containing all threespecies of Bordetella. If a biological sample contains only B.pertussis, only the first amplification product will be produced as nonucleotide sequences are present for SEQ ID NOS: 4 and 5 or 7 and 8 tohybridize to. Similarly, if a biological sample contains a co-infectionwith B. pertussis and B. parapertussis, a first and a thirdamplification product will be produced and a second amplificationproduct (illustratively specific to B. holmesii) will be absent. Thus,the presence of the first and third amplification products will signal aco-infection with B. pertussis and B. parapertussis.

It is appreciated that the IS481 sequence amplified when primers SEQ IDNO: 1 and SEQ ID NO: 2 are used will produce an amplification productwith either a B. pertussis or a B. holmesii infection. Thus, when abiological sample contains a co-infection of B. pertussis and B.holmesii a first amplification product will be produced. Similarly, in abiplex or triplex assay where primers SEQ ID NOS: 4 and 5 are used inaddition to SEQ ID NOS: 1 and 2, two amplification products will beobserved indicating the presence of a co-infection of B. holmesii and B.pertussis or possibly a single infection of B. holmesii.

However, to date no reports of a co-infection of B. pertussis and B.holmesii in a human population has been reported or observed in studiesof a large number of biological samples from patients suspected ofhaving a Bordetella infection. It is not expected that thiscross-reactivity will be detrimental to clinical analysis, particularlywhen the inventive triplex assay is employed in conjunction with theinventive ptxS1 assay describe ante.

The ptxS1 assay capitalizes on the unconventional search for anucleotide sequence that will detect multiple Bordetella species. (SeeTatti, K M, et al., Diag. Micro. Infec. Dis., 2008; 61:264-271 thecontents of which are fully incorporated herein by reference,particularly for description of ptxS1 and IS481 amplifications.)Specifically, the inventor sought to locate a nucleotide sequence thatis specific for both B. pertussis and B. parapertussis, but is absentfrom B. holmesii. Primers 5′-CGCCAGCTCGTACTTC-3′ (SEQ ID NO: 10) and5′-GATACGGCCGGCATT-3′ (SEQ ID NO: 11) are employed to amplify a sequencethat will not produce a reaction product indicative of B. holmesii.Thus, in the above triplex inventive assay should (first) primers SEQ IDNOS: 1 and 2 and (second) primers SEQ ID NOS: 4 and 5 each produce afirst and second amplification product, a parallel or subsequentanalysis by the ptxS1 analysis will identify whether there is a dualinfection of B. pertussis and B. holmesii or a single infection of B.holmesii. A positive ptxS1 assay will indicate a co-infection whereas anegative ptxS1 assay will indicate a single infection with B. holmesii.

The inventive ptxS1 assay is similarly operable in a real-time PCRassay. A probe sequence that recognizes the amplification product of theptxS1 nucleic acid sequence is operable to detect increasing copy numberof the amplification product. Preferably, a probe sequence is5′-AATACGTCGACACTTATGGCGA-3′ (SEQ ID NO: 12).

Optionally, a quadraplex assay is performed where four matched primersets are simultaneously used to analyze a biological sample for IS481,hIS1001, pIS1001, and ptxS1. This assay system is operable without theneed for a second confirmatory ptxS1 assay. However, as clinicallaboratories are continually seeking the least cost solution and sinceno co-infection of B. holmesii and B. pertussis has been observed andreported, the need for quadraplex assay will be user specific.

In a specific embodiment, the processes further involve obtaining acontrol sample from a control subject, contacting the control samplewith a compound or agent capable of detecting the presence of Bordetellanucleic acid in the sample, and comparing the presence of mRNA orgenomic DNA in the control sample with the presence of mRNA or genomicDNA in the test sample.

The invention also encompasses kits for detecting the presence ofBordetella nucleic acid sequences in a test sample. The kit, forexample, includes a labeled compound or agent capable of detecting anucleic acid molecule in a test sample and, in certain embodiments, fordetermining the titer in the sample.

For oligonucleotide-based kits, the kit includes, for example: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, thathybridizes to a nucleic acid sequence of the Bordetella species ofinterest and/or (2) a pair of primers (one forward and one reverse)useful for amplifying a nucleic acid molecule containing the Bordetellasequence. The kit can also comprise ancillary agents, e.g., a bufferingagent, a preservative, or a protein stabilizing agent. The kit can alsocomprise components necessary for detecting the detectable agent (e.g.,an enzyme or a substrate). The kit can also contain a control sample ora series of control samples which is assayed and compared to the testsample contained. Each component of the kit is usually enclosed withinan individual container and all of the various containers are usuallyenclosed within a single package along with instructions for use.

The instant inventive processes are amenable to use for diagnosis ofBordetella infection in a subject, insects, and any inclusive otherorganism capable of infection or transfection by or with Bordetella.

To increase confidence and to serve as an internal or external control,a purified and titered Bordetella solution is used as a biologicalsample. It is appreciated that any target Bordetella species is operableas a control. Illustratively, a control sample includes B. pertussis, B.holmesii, B. parapertussis, or combinations thereof. By amplification ofa single sample with known quantities of Bordetella or of a set ofsamples representing a titration of Bordetella, the level of Bordetellain the unknown biological sample is determined. Preferably, the purifiedand titered Bordetella solution is analyzed in parallel with the unknownbiological sample to reduce inter assay error or to serve as a standardcurve for quantitation of unknown Bordetella in the biological sample.Using purified and titered Bordetella solution provides for a similarcomplete genetic base DNA strand for amplification.

In another embodiment, a subgenomic fragment is cloned into a plasmidfor amplification, purification, and use as a quantitative comparator ornucleic acid calibrator. In a non-limiting example, a DNA subgenomicfragment of B. pertussis is optionally amplified from a positive nasalswab using primers bracketing the PCR target regions in the IS481sequence. It is appreciated that other sequences are similarly suitablefor use as a quantitative control. The known concentration of thesubgenomic fragment is used to create a standard curve for quantitativedeterminations and to access amplification efficiency.

Also provided is a kit for detecting Bordetella infection that containsreagents for the amplification, or direct detection of Bordetella orportions thereof. An exemplary kit illustratively includes a forward andreverse primer pair, a non-degenerate probe. In a preferred embodiment,the forward and reverse primers have the oligonucleotide sequence SEQ IDNOS: 1 and 2 and a nondegenerate probe of the sequence SEQ ID NO: 3. Itis appreciated that a diagnostic kit may optionally contain primers andprobes that are the complements of SEQ ID NOS 1-3 or that hybridize witholigonucleotides SEQ ID NOS: 1-3. It is further appreciated that adiagnostic kit optionally includes ancillary reagents such as buffers,solvents, thermostable polymerases, nucleotides, and other reagentsnecessary and recognized in the art for amplification and detection ofBordetella in a biological sample.

In a preferred embodiment a kit includes reagents for detection anddistinguishing three or more species of Bordetella. Preferably, a kitincludes reagents for detection and distinguishing B. pertussis, B.holmesii, and B. parapertussis. Most preferably, a kit includes primersand probes of SEQ ID NOS: 1-9. Optionally, a kit may also includereagents of SEQ ID NOS: 10-12.

The invention provides a host cell containing a nucleic acid sequencesaccording to the invention as an alternative to synthetic primersequence generation. Plasmids containing the polymerase components ofthe Bordetella bacteria are generated in prokaryotic cells for theexpression of the components in relevant cell types (bacteria, insectcells, eukaryotic cells). Preferably, the cell line is a primate cellline. These cell lines may be cultured and maintained using known cellculture techniques such as described in Celis, Julio, ed., 1994, CellBiology Laboratory Handbook, Academic Press, NY. Various culturingconditions for these cells, including media formulations with regard tospecific nutrients, oxygen, tension, carbon dioxide and reduced serumlevels, can be selected and optimized by one of skill in the art.

The preferred cell line of the present invention is a prokaryotic cellline such as E. coli and the like for transiently or stably expressingone or more full-length or partial Bordetella proteins. Such cells canbe made by transfection (proteins or nucleic acid vectors), infection(viral vectors) or transduction (viral vectors). The cell lines for usein the present invention are cloned using known cell culture techniquesfamiliar to one skilled in the art. The cells are cultured and expandedfrom a single cell using commercially available culture media underknown conditions suitable for propagating cells.

A host cell is a cell derived from a mammal, insect, yeast, bacteria, orany other single or multicellular organism recognized in the art. Hostcells are optionally primary cells or immortalized derivative cells.Immortalized cells are those which can be maintained in vitro forseveral replication passages.

Methods involving conventional biological techniques are describedherein. Such techniques are generally known in the art and are describedin detail in methodology treatises such as Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, NY, 1992 (with periodic updates). Immunologicalmethods (e.g., preparation of antigen-specific antibodies,immunoprecipitation, and immunoblotting) are described, e.g., in CurrentProtocols in Immunology, ed. Coligan et al., John Wiley & Sons, NY,1991; and Methods of Immunological Analysis, ed. Masseyeff et al., JohnWiley & Sons, NY, 1992. Additionally, numerous techniques and reagentsoperable herein are described in Tatti, K M, et al., Diag. Micro. Infec.Dis., 2008; 61:264-271.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.While the examples are generally directed to mammalian cells, tissue,fluids, or subjects, a person having ordinary skill in the artrecognizes that similar techniques and other techniques know in the artreadily translate the examples to other mammals such as humans. Reagentsillustrated herein are commonly cross reactive between mammalian speciesor alternative reagents with similar properties are commerciallyavailable, and a person of ordinary skill in the art readily understandswhere such reagents may be obtained.

EXAMPLE 1 Obtaining Bacterial Strains and Clinical Specimens

Bacterial strains are obtained from the CDC culture collections in theMeningitis and Vaccine Preventable Diseases Branch/Pertussis andDiphtheria Laboratory and other collaborators at CDC. Two hundredfifty-eight Bordetella spp. isolates are grown for 4 days at 37° C.under high humidity on modified Regan-Lowe medium containing charcoalagar (Oxoid, Basingstoke, UK) and 10% defibrinated horse blood. Otherisolates were cultured following standard procedures.

A total of 101 clinical human specimens of nasopharyngeal aspirates orswabs were submitted to CDC during various cough-illness outbreaks (CDC,2004, 2006, 2007). The specimens were cultured for Bordetella spp. uponarrival as described by Hardwick et al., 2002. In accordance with CDChuman subjects guidelines, this study is considered exempt ofinstitutional review board approval because the linkage to personallyidentifiable information is not accessible to CDC investigators.

EXAMPLE 2 Preparation of Nucleic Acid and Sequencing of BacterialStrains

DNA extraction from clinical specimens or isolates is performed eitheron MagNA Pure LC equipment using MagNA Pure LC DNA Isolation Kit III(Roche Applied Science, Indianapolis, Ind.) or using the QIAampDNA MiniKit (QIAGEN, Valencia, Calif.) following the manufacturers' instructionsand eluted in 100 μL of either 10 mmol/L Tris-HCl buffer (pH 8.0) orwater. DNA is extracted from clinical specimens obtained when theoutbreaks occurred. Concentration of DNA extracted from isolates isdetermined with a NanoDrop® ND-1000 spectrophotometer (NanoDropTechnologies, Wilmington, Del.).

EXAMPLE 3 Preparation of Primers and Probes

The primers and probes are designed based on the nucleotide sequences ofB. pertussis IS481 (GenBank Accession No. M28220), B. holmesii (GenBankAccession No. AY786982), B. parapertussis (GenBank Accession No.X66858), and ptxS1 (GenBank Accession No. M14378) using Primer Express2.0 or 3.0 (Applied Biosystems, Foster City, Calif.), and analyzed usingOligo 6.0 (Molecular Biology Insights, Cascade, Colo.). Primers andprobes are synthesized in the Biotechnology Core Facility at CDC usingper solid support techniques known in the art (Table 1).

EXAMPLE 4 Real-time PCR to Identify and Distinguish Bordetella Species

Real-time PCR assays are performed on either a LightCycler or AB7500instrument. Real-time PCR with the AB7500 instrument (AppliedBiosystems) is performed in a total volume of 25 μL in an optical96-well plate (Applied Biosystems). The amplification mixture contains 4μL of extracted DNA plus a 21-μL TaqMan® Gene Expression PCR Master Mix(Applied Biosystems) with 100 nmol/L of each primer and 300 nmol/L ofprobe for IS481 target, 100 nmol/L of each primer and 100 nmol/L ofprobe for hIS1001 target, and 300 nmol/L of each primer and 100 nmol/Lof probe for pIS1001 target. A similar assay mix is used for the ptxS1target that contains 700 nmol/L of each primer and 300 nmol/L probe. ThePCR protocol used is as follows: hold for 2 min at 50° C.; enzymeactivation for 10 min at 95° C.; amplification for 15 s at 95° C. and 1min at 60° C., repeat for 45 cycles. The PCR protocol for the ptxS1assay was the same as for the triplex assay, except the annealingtemperature is 57° C. Positive samples (strain A639) and nontemplatecontrol samples (PCR grade water) are tested in triplicate in each run.

EXAMPLE 5 Assay Sensitivity

Assay sensitivity is determined by comparison of serial dilutions ofrepresentative Bordetella strains. A singleplex assay for eachindividual real-time PCR target consisting of a 10-fold serial dilutionis run simultaneously with the multiplex assay for a comparison of cyclethreshold (Ct) values. Sensitivity is measured using serially diluted B.pertussis, B. parapertussis, and B. holmesii isolates. Bacterialcultures are suspended in 10 mmol/L tris-buffered saline (TBS) (pH 7.2)at a concentration equivalent to 10⁷ cells/mL based on McFarlandturbidometric standards. Serial 10-fold dilutions (10⁷ to 10⁰ CFU/mL)are made from the suspension in TBS. Aliquots from each series ofdilutions are used to spread plates in triplicate, and the averagecolony-forming unit per milliliter is calculated. DNA is extracted fromthe aliquots and tested in the real-time PCR assays in triplicate todetermine the linear dynamic range and lower limit of detection (LLOD).A model of nonlinear regression (Sigma Plot version 9.0) is used for thedynamic range analysis, and the regression line represents data in thelinear range. The PCR efficiency (E) of the primer pair and probe iscalculated using the equation E=10⁻¹/slope⁻¹ (Vaerman et al., 2004). AnE of 1.0 indicates that the amplicon quantity is duplicated every cycle(FIG. 1).

A stock concentration of 25 ng/μL of DNA from B. pertussis strain A639is determined based on the absorption at A260, and 10-fold serialdilutions are tested in triplicate in the real-time PCR assays todetermine the lower limit of detection (LLOD) per PCR reaction based ongenome equivalents for both the LightCycler and AB7500 assays.

Each individual target assay achieves >99% efficiency with linearamplification over a 5-log dynamic range. The PCR efficiency (E) of eachprimer pair and probe set is near a value of 1 indicating that theamplicon quantity is duplicated every cycle and demonstrates anexponential amplification of DNA with each of the primer and probe sets.The IS481 assay achieves >99% efficiency with linear amplification overan 8-log dynamic range of 10⁰ to 10⁷ CFU/mL for B. pertussis (FIG. 1A),whereas the ptxS1 assay achieves >99% efficiency with linearamplification over a 6-log dynamic range of 10² to 10⁷ CFU/mL for B.pertussis (FIG. 1B). The regression coefficients (R²=0.996 and 0.997)and the amplification efficiencies (E=1.0105 and 1.009) for the IS481and ptxS1 assays, respectively, demonstrated an exponentialamplification of DNA with both primer and probe sets.

The multiplex assay demonstrates an amplification reaction patternsimilar to a singleplex assay of each target (FIG. 2). The multiplexR-PCR assay increases the Ct values for each target slightly resultingin 1 genomic equivalent per reaction for B. holmesii IS/001-like(hIS1001) and IS481 but remains less than 1 genomic equivalent perreaction for the B. pertussis IS481 and the B. parapertussis pIS1001targets (Table 2).

TABLE 2 Comparisons between single target real-time PCR assay andmultiplex real-time PCR assays. B. pertussis DNA with FAM labeled IS481Comparison between assays Genomic Singleplex Assay Multiplex AssayEquivalents 300 nM/300 nM 100 nM/300 nM 10,000 16.76 17.22 1,000 19.4320.06 100 23.02 23.50 10 26.35 27.39 1 30.77 30.55 0.1 33.52 34.21 B.holmesii DNA with FAM labeled IS481 Comparison between assays GenomicSingleplex Assay Multiplex Assay Equivalents 100 nM/100 nM 100 nM/100 nM10,000 21.54 22.21 1,000 25.66 26.19 100 29.36 30.10 10 33.75 34.08 137.06 37.53 0.1 42.21 40.00 B. holmesii DNA with IS1001 Quasar 670labeled Comparison between assays Genomic Singleplex Assay MultiplexAssay Equivalents 100 nM/100 nM 100 nM/100 nM 10,000 22.12 22.62 1,00025.69 26.13 100 29.38 29.80 10 33.74 34.01 1 38.11 37.61 0.1 41.07 40.82B. parapertussis DNA with 5′Hex labeled IS1001 Comparison between assaysGenomic Singleplex Assay Multiplex Assay Equivalents 300 nM/100 nM 300nM/100 nM 10,000 19.20 19.28 1,000 22.57 22.54 100 26.26 26.36 10 29.9830.42 1 33.63 33.48 0.1 35.97 36.60

EXAMPLE 6 Specificity of Targets for B. Pertussis, B. Holmesii, and B.Parapertussis

Prior to the multiplex assay, a total of 258 Bordetella species isolateswhich includes 60 B. pertussis, 52 B. parapertussis, 70 B.bronchiseptica, and 72 B. holmesii are used in the individual evaluationof each real-time PCR target assay for cross-reactivity at a 5 ng/μlconcentration. One isolate each of B. avium, B. hinzii, B. petrii, andB. trematum are also tested. All isolates are acquired from a humanreservoir. The IS481 target sequence is completely specific for the B.pertussis and B. holmesii isolates. The hIS1001 target is specific to B.

TABLE 3 Specificity analyses No. Genus Species tested Aerococcus A.viridans 1 Bacillus B. cereus, B. subtilis 2 Chlamydia C. pneumoniae 1Corynebacterium C. diphtheriae, C. ulcerans, C. 8 accolens, C. jeikeium,C.minutissimum, C. pseudodiphtheriticum,C. pseudotuberculosis, C.striatum Enterococcus E. faecalis 1 Escherichia E. coli 1 FlavobacteriumF. meningosepticum 1 Gemella G. haemolysans 1 Haemophilus H. influenzaeserotype, a, b, c, d, e, f, NT^(a), H. 10 haemolyticus, H. aegyptius, H.parainfluenzae Legionella L. pneumophila, L. longbeachae serogroup 3 1and 2 Moraxella M. catarrhalis 1 Mycoplasma M. pneumoniae 1 Neisseria N.meningitidis serogroup, A, B, C, W135, X, 13 Y, Z, 29E, NG^(b), N.sicca, N. lactamica, N. subflava, N. cinerea Pseudomonas P. aeruginosa 1Staphylococcus S. aureus 1 Streptococcus S. pneumoniae, S.agalactiae(2), S. pyogenes, 20 S. canis, S .anginosus, S. equi, S.zooepidemicus, S. porcinus, S. dysgalactiae(2), S. constellatus, S.iniae, S. intermedius, S. bovis, S. pseudopneumoniae,S. mitis, S.oralis, S. sanguinis, S. salivarius Total 66holmesii. The pIS1001 target is specific to B. parapertussis and showsno cross-reactivity with B. pertussis or B. holmesii, but reacts with 5of 72 B. bronchiseptica isolates, however, B. bronchiseptica is expectedto be of little clinical relevance to a human population.

A collection of non-Bordetella spp. (n=66) listed below (Table 3) andhuman DNA are also tested for cross-reactivity with each individualreal-time PCR target. No cross-reactivity of the primer/probe sets isobserved with DNA from these species.

EXAMPLE 7 Detection and Distinguishing Bordetella Species in ClinicalSpecimens

In the blinded retrospective study, 101 nasopharyngeal specimenscollected in cough-illness outbreaks were tested using the multiplex andsingleplex assays.

DNA extracted from nasopharyngeal specimens is assayed in triplicatewith a water control placed between every 2 samples. An average cyclethreshold (Ct) value of the multiplex PCR assays was calculated to givea final value. If a specimen is positive in 2 of 3 tests, it isconsidered positive. Clinical specimens are also tested for the humanrnaseP gene using a real-time PCR assay to monitor the quality of DNA inthe specimen and to check for inhibition as described by Tatti, K M, etal., Diag. Micro. Infec. Dis., 2008; 61:264-271. To be consideredpositive for rnaseP, a specimen should have a Ct value<40. If a Ct valueis greater than 40 or negative for rnaseP, a 1- to 5-fold dilution ofthe specimen in water is performed, and the multiplex assays arerepeated on the diluted sample. The rnaseP results demonstratedamplifiable DNA without PCR inhibitors in all specimens. The range of Ctvalues for rnaseP was from 21.7 to 35.0.

A total of 24.7% of the clinical specimens were positive for Bordetellaspp. by culture, whereas 30.7% were positive for Bordetella spp. by PCRsupporting the accuracy of the inventive assay. Moreover, all 37specimens that generated PCR amplicons gave comparable Ct values in boththe singleplex and the multiplex assays. Of the 37 clinical specimensthat generated Ct values, 6 specimens had 2 of 3 replicates with a highCt value (Ct≧35) with IS481 alone and were interpreted as indeterminate.Sixty-four (63.4%) clinical specimens were culture and PCR negative.

Twenty-seven (26.7%) of the samples were positive for B. pertussis byreal-time PCR demonstrating amplification of IS481, but no amplificationof either hIS1001 or pIS1001.

Two clinical specimens were culture positive for B. parapertussis andPCR positive for both B. pertussis by IS481 and B. parapertussis bypIS1001. The results from the multiplex assay demonstrate that thespecimens are in fact co-infections of the two species. This exemplifiesthe robustness of the multiplex assay.

Two samples were positive for B. holmesii by hIS1001 in the multiplexreal-time PCR assay and negative by the ptxS1 assay which demonstratesinfection only by B. holmesii and not B. pertussis. These resultssuggest that co-infection of B. holmesii and B. pertussis is extremelyrare.

EXAMPLE 8 Detection of Bordetella Amplicons Via Mass Spectroscopy

Detection of amplification products obtained as in Example 4 wasperformed essentially as described by Blyn, L, et al. J. Clin.Microbiol. 2008; 46(2):644-651. Following amplification each PCR mixtureis desalted and purified using a weak anion-exchange protocol based onthe method of Jiang and Hofstadler (Jiang, Y, and S A Hofstadler. Anal.Biochem. 2003; 316:50-57). ESI-TOF is used to obtain accurate-mass (±1ppm), high-resolution (M/ΔM, >10,000 full width half maximum) massspectra. For each sample, approximately 1.5 μl of analyte solution isconsumed during the spectral acquisition. Raw mass spectra arepostcalibrated with an internal mass standard and deconvolved to averagemolecular masses. Quantitative results are obtained by comparing thepeak heights with an internal PCR calibration standard present in everyPCR well at 300 molecules unless otherwise indicated. This assayconfirms the data obtained as in Example 7.

Patent applications and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. The foregoing description is illustrative ofparticular embodiments of the invention, but is not meant to be alimitation upon the practice thereof. The following claims, includingall equivalents thereof, are intended to define the scope of theinvention.

The invention is hereby described with relation to the followingreferences and those otherwise identified in the instant specification.Each reference is incorporated herein by reference as if each were laidout explicitly in its entirety in the instant specification includingboth text and figures. Each reference is incorporated for the individualpoint referred to in the specification as well as for all informationcontained therein and not explicitly identified in the specification.All references are representative of the knowledge of a person of skillin the art and illustrate other aspects of the present invention asenvisioned by the inventors.

The invention claimed is:
 1. A process of detecting and distinguishingBordetella pertussis, B. parapertussis, and B. holmesii in a biologicalsample comprising: producing a first amplification product by amplifyinga Bordetella nucleotide sequence if present in the sample using aforward primer consisting of the sequence of SEQ ID NO: 1 and a reverseprimer consisting of the sequence of SEQ ID NO: 2, under conditionssuitable for a polymerase chain reaction; measuring said firstamplification product; producing a second amplification product byamplifying a Bordetella nucleotide sequence if present in the sampleusing a forward primer consisting of the sequence of SEQ ID NO: 4 and areverse primer consisting of the sequence of SEQ ID NO: 5, underconditions suitable for a polymerase chain reaction, and measuring saidsecond amplification product; producing a third amplification product byamplifying a Bordetella nucleotide sequence if present in the sampleusing a forward primer consisting of the sequence of SEQ ID NO: 7 and areverse primer consisting of the sequence of SEQ ID NO: 8, underconditions suitable for a polymerase chain reaction, and measuring saidthird amplification product; said step of producing said firstamplification product, said step of producing said second amplificationproduct, and said step of producing said third amplification productperformed in the same reaction chamber; producing a fourth amplificationproduct by amplifying a Bordetella nucleotide sequence using a forwardprimer consisting of the sequence of SEQ ID NO: 10 and a reverse primerconsisting of the sequence of SEQ ID NO: 11, under conditions suitablefor a polymerase chain reaction, and measuring said fourth amplificationproduct; detecting and distinguishing Bordetella pertussis, B Bordetellaparapertussis, and B Bordetella holmesii in the sample based on arelative ratio of said first amplification product, said secondamplification product, said third amplification product, and said fourthamplification product.
 2. The process of claim 1 wherein measuring oneor more of said first amplification product, said second amplificationproduct, and said third amplification product uses as a probe consistingof the sequence of SEQ ID NO: 3 and a label and a quencher, SEQ ID NO: 6and a label and a quencher, SEQ ID NO: 9 and a label and a quencher, orcombinations of said probes as a detection probe, wherein one or more ofsaid probes is hybridized to one or more of said amplification products;and detecting the presence or absence of a first detection signal, asecond detection signal and/or a third detection signal from one or moreof said probes.
 3. The process of claim 1 further comprising providing astrain-specific B. holmesii negative probe consisting of the sequence ofSEQ ID NO: 12 and a label and a quencher; hybridizing saidstrain-specific probe under conditions suitable for a polymerase chainreaction to said complementary fourth amplification product to produce afourth detection signal; and detecting the presence or absence of saiddetection signal from said probe of SEQ ID NO:
 12. 4. The process ofclaim 3, wherein said first, second, third, or fourth detection signalis compared to a fifth detection signal from a nucleic acid calibratorextracted in parallel to said biological sample.
 5. The process of claim4, wherein said nucleic acid calibrator comprises a known amount of aBordetella bacteria and a known amount of a medium.
 6. The process ofclaim 4 wherein said fifth detection signal is generated by PCRamplification of a titered Bordetella solution.
 7. The process of claim1 wherein said detecting diagnoses Bordetella infection.
 8. The processof claim 1 wherein said fourth amplification product is generated inparallel with said first amplification product.
 9. The process of claim1 wherein said detecting is by gel electrophoresis, Southern blotting,liquid chromatography, mass spectrometry, liquid chromatography/massspectrometry, static fluorescence, dynamic fluorescence, highperformance liquid chromatography, ultra-high performance liquidchromatography, enzyme-linked immunoadsorbent assay, real-time PCR,RT-PCR, nucleotide sequencing, or combinations thereof.